Benzopyran derivatives having inhibitory activities against liver fibrosis and cirrhosis and their pharmaceutical uses

ABSTRACT

The present invention relates to a novel benzopyran derivative having goodantagonistic activity on TGF-β receptor which can be effectively used as a prophylactic and therapeutic agent for liver disease as well as several fibroplasiadiseases such as hepatic fibrosis, liver cirrhosis, pulmonary fibrosis, dermatosclerosis, glomerular fibrosis and the like; and a pharmaceutical use thereof.

This application is a 371 of PCT/KR2005/001833 filed on Jun. 15, 2005,published on Jul. 6, 2006 under publication number WO 2006/070984 A1which claims priority benefits from Korean Patent Application No.10-2004-0117711 filed Dec. 31, 2004 and Korean Patent Application No.10-2004-0117707 filed Dec. 31, 2004, the disclosures of which are herebyincorporated by reference.

FIELD OF THE INVENTION

The present invention relates to a novel benzopyran derivative havinggood antagonistic activity on TGF-β receptor which can be effectivelyused both as a prophylactic and a therapeutic agent for treating liverdisease as well as several fibroplasia diseases such as hepaticfibrosis, liver cirrhosis, pulmonary fibrosis, dermatosclerosis,glomerular fibrosis and so on; and a pharmaceutical use thereof.

BACKGROUND OF THE INVENTION

Liver plays an important role of metabolizing exogenous and endogenoussubstances in human body. When liver tissues are continuously exposed tofrequent alcoholic drinking or crapulence, viral infection or drugmisuse and abuse, the liver tissues are easily damaged and subsequentlydevelop into chronic liver diseases such as hepatic fibrosis and livercirrhosis. Since hepatic fibrosis does not show any pain or a particularsymptom until it is discovered at a terminal stage, its death rate isrelatively high and thus its development often becomes a social problem.

In particular, liver disease can be divided into several proceduralsymptoms. The damaged tissues are first converted into fatty liver inits early stage and then developed into hepatitis, and finally intohepatic fibrosis and liver cirrhosis. Generally, it has been reportedthat the progress up to hepatic fibrosis is reversible, but once it isdeveloped into liver cirrhosis, its subsequent process becomesirreversible. Accordingly, liver disease can be cured by administering adrug at a stage prior to the development of hepatic fibrosis or an earlyfibrosis stage.

Liver cirrhosis is caused by fibrosis of liver tissues. Liver fibrosisis a condition that the balance between the synthesis and degradationprocedures of a connective tissue is lost, caused by excessiveaccumulation of connective tissues within liver tissues, and accompaniedby necrosis or inflammation. In particular, hepatic stellate cells(HSCs) that store vitamin A in normal liver are converted intomyofibroblasts by acute and chronic liver damage, rapidly proliferateand synthesize an excessive amount of connective tissues through theincrease in the synthesis and translocation of an extracellular matrixsuch as collagen, proteoglycan or hyaluronan, which results instimulating the progression of liver fibrosis [Friedman et al., Proc.Natl. Acad. Sci. USA., 82: 8681 (1985) Gressner et al., Biochem.Biophys. Res. Commun., 151: 222 (1988) Gressner et al., J. Hepatol., 22:28 (1995)]. In such procedure, TGF-β is synthesized, secreted andactivated by several kinds of cells in the liver tissues, in particular,Kupffer cells or hepatic stellate cells activated by TGF-β, induce theproliferation and development of hepatic stellate cells, and thereby,play an important role in inducing the over-production and accumulationof an extracellular matrix such as collagen. It has been reported thatin chronic liver disease such as hepatic fibrosis or liver cirrhosis,TGF-β is only expressed in the liver tissues undergoing fibrosis,increases the amount of an extracellular matrix, and finally, stimulatesthe progression of hepatic fibrosis [Bauer and Schuppan, FEBS Lett.502:1-3 (2001); Bedossa and Paradis, J Hepatol., 22 (Suppl. 2):37-4(1995)].

Until now, the development of an inhibitor for hepatic fibrosis or atherapeutic agent for liver cirrhosis has been focused on thedevelopment of a drug which inhibits the over-production of a connectivetissue (representatively, collagen) of hepatic stellate cells orinhibits the growth thereof, but it has not yet been developed as aneffective therapeutic agent. Recently, studies have been activelyconducted on the inhibition of TGF-β function or TGF-β receptoractivation as a target for developing a new therapeutic agent for livercirrhosis, based on the fact that TGF-β is the strongest induciblefactor for fibrosis of hepatic stellate cells among cytokines involvedin fibrosis.

The present inventors have therefore synthesized numerous compoundshaving antagonistic activity on TGF-β receptor, endeavored to screen anew compound capable of inhibiting or preventing hepatic fibrosis amongthem by employing hepatic stellate cells that play an important role inthe progression of hepatic fibrosis, and finally, found that abenzopyran derivative having a novel structure shows significantprophylactic and therapeutic effect.

DETAILED DESCRIPTION OF THE INVENTION

The present inventors have synthesized various benzopyran derivativesbased on the fact that natural and synthetic substances having abenzopyran backbone show a pharmacological efficacy broadly suppressingactive oxygen, and studied their antagonistic activities for TGF-βreceptor which strongly induces fibrosis of hepatic stellate cells, andthe inhibition of collagen synthesis and cell proliferation by usinghepatic stellate cells which are known as a major cause of hepaticfibrosis. As a result, it is discovered now that a thiourea- or aguanidine-based benzopyran derivative shows an inhibitory effect onhepatic fibrosis through antagonistic activity on TGF-β receptor and theinhibition of collagen synthesis.

Accordingly, in an embodiment of the present invention, there isprovided a novel benzopyran derivative useful for developing atherapeutic agent for liver cirrhosis and a method for its preparation.

In another embodiment of the present invention, there is provided a useof the novel benzopyran derivative as both a prophylactic and atherapeutic agent for liver diseases caused by the activation andover-production of TGF-β and the excessive accumulation of anextracellular matrix such as collagen.

In a further embodiment of the present invention, there is provided abenzopyran derivative described in Formula (1):

wherein Y is S or N—R⁴;

R¹ and R⁴ are independently C₁-C₂₀ alkyl, amine, substituted orunsubstituted phenyl, substituted or unsubstituted benzyl, dioxobenzyl,isovaline(methylester), naphtyl, or phenyl-X— (wherein X is carbonyl, orC₁-C₆ alkyl), or R¹ and R⁴ are fused together with the nitrogen atom towhich they are attached to form a heterocycle having a 5- to 7-memberring;

R² is hydrogen, or C₁-C₅ alkyl;

R³ is hydrogen, C₁-C₅ alkyl, substituted or unsubstituted phenyl, orsubstituted or unsubstituted benzyl; and

wherein the substituted phenyl or the substituted benzyl is phenyl orbenzyl replaced with 1 to 4 substituents selected from the groupconsisting of halogen, nitro, benzyloxy, C₁-C₅ alkyl, C₁-C₅ alkoxy,C₁-C₅ haloalkyl, C₁-C₅ alkylsulfide, and C₁-C₅ alkylsulfanyl.

Further, since in case of presenting a different substituent at theposition 2 of benzopyran in the benzopyran derivative of Formula (1) inaccordance with the present invention, the benzopyran derivative has anoptical activity, isomers of the compound of Formula (1) may be includedin the scope of the present invention.

Of the compounds of Formula (1) according to the present invention, thepreferred ones are:

those wherein R¹ and R⁴ are independently C₁-C₂₀ straight, branched andcyclic alkyl; amine; phenyl; phenyl replaced with 1 to 4 substituentsselected from the group consisting of halogen, nitro, benzyloxy, C₁-C₅alkyl, C₁-C₅ alkoxy, C₁-C₅ haloalkyl, C₁-C₅ alkylsulfide, and C₁-C₅alkylsulfanyl; benzyl; benzyl replaced with halogen; dioxobenzyl;isovaline(methylester); morphorino; naphtyl; or R¹ and R⁴ are fusedtogether with the nitrogen atom to which they are attached to formpiperidine, piperidine replaced with C₁-C₅ alkoxycarbonyl, piperazine,or piperazine replaced with phenyl;

R² is hydrogen, or C₁-C₅ alkyl; and

R³ is hydrogen, C₁-C₅ alkyl, phenyl, or benzyl.

In a still further embodiment of the present invention, there isprovided a method for preparing the compound of Formula (1) by using aliquid phase high-throughput synthetic technique, which is described inReaction Scheme 1 as follows:

wherein R¹, R², R³, and R⁴ have the same meanings as defined in Formula(1).

6-Amino-2,7-disubstituted-2-methyl-2H-chromen of Formula (2) used as astarting material in the preparation method of the present invention isa known compound, which can be easily synthesized according to aconventional method in the art.

The preparation method of the present invention in accordance withReaction Scheme 1 comprises: (1) synthesizing a thiourea-basedbenzopyran derivative of Formula (1a) by reacting6-Amino-2,7-disubstituted-2-methyl-2H-chromen of Formula (2) with anisothiocyanate derivative of Formula (3); and (2) synthesizing aguanidine-based benzopyran derivative of Formula (1b) by reacting thethiourea-based benzopyran derivative of Formula (1a) with an aminederivative of Formula (5).

Further, the preparation method of the present invention ischaracterized by the step of removing the unreacted isothiocyanate ofFormula (3) after the step (1) is completed and the unreacted aminederivative of Formula (5) after the step (2) is completed by filtrationusing a scavenger resin, respectively.

Namely, the method of the present invention can synthesize a largeamount of the thiourea-based benzopyran derivative at the same timewithin a relatively short period of time while removing the unreactedisothiocyanate derivative of Formula (3) after the step (1) is completedby filtration using the scavenger resin having an amine group of Formula(4):

wherein {circle around (P)} is a solid support in the form of a polymerselected from the group consisting of polystyrene,polystyrene-divinylbenzene, polymethacrylic acid-dimethylacrylamide andpolyhydroxy methacrylic acid.

Further, since the method of the present invention can purify a largeamount of reactants simultaneously while removing the unreacted aminederivative of Formula (5) after the step (2) is completed by filtrationusing the scavenger resin containing an isocyanate group of Formula (6),it is capable of synthesizing the guanidine-based benzopyran derivativewithin a short period:

wherein {circle around (P)} is a solid support in the form of a polymerselected from the group consisting of polystyrene,polystyrene-divinylbenzene, polymethacrylic acid-dimethylacrylamide andpolyhydroxy methacrylic acid.

A reaction process, composition of a solvent system and selectable rangeof a reaction condition in accordance with the present invention aredescribed in more detail as follows. For the purpose of conducting thepreparation method of the present invention, it is desirable to use asolvent having a high swelling effect of a resin considering the use ofa scavenger resin at a final step. In particular, the solvent employablein the present invention includes dichloromethane (CH₂Cl₂), chloroform(CHCl₃), tetrahydrofuran (THF) and the like. In the above step (1), inorder to incorporate R¹ substitutent, it is preferable to use theisocyanate derivative replaced with R¹ of Formula (3) in the amountranging from 1.2 to 2.0 equivalents, and preferably, the use of 1.2equivalents is more economical. In the step (2), it is preferable to usethe amine derivative replaced with R² of Formula (5) in the amountranging from 1.2 to 2.0 equivalents based on the amount of thiounreabased benzopyran derivative of Formula (1a), and it is more preferableto use 1.2 equivalents of the amine derivative in terms ofcost-effectiveness.

Further, in order to confirm the production of a target compound in thecourse of carrying out the preparation method of the present invention,the final reactant may be separated and purified by using ahigh-throughput multiple column chromatography (Quad³⁺; Biotage, USA)and automatic sample injector at a final step, and then, subjected to astructural analysis with NMR and Mass spectra.

Furthermore, since 6-Amino-2,7-disubstituted-2-methyl-2H-chromen ofFormula (2) used as a starting material and a target product, banzopyranderivatives of Formula (1) in the preparation method according to thepresent invention have their optical isomers, respectively, each oftheir pure optical isomer compounds may be separated by means of aconventional purification method well-known in the art upon necessity.

Meanwhile, the compound of the present invention can be used as aneffective prophylactic and therapeutic agent for treating various liverdiseases caused by TGF-β activity, collagen synthesis activity, andliver cirrhosis activity. Accordingly, the present invention includes aprophylactic as well as a therapeutic agent for treating liver diseasescomprising the benzopyran derivative of Formula (1) or apharmaceutically acceptable salt thereof as an active ingredient.Further, since the compound in accordance with the present invention hashigh antagonistic activity on TGF-β receptor, it can be also used forpreventing and treating various fibroplasia diseases such as hepaticfibrosis, liver cirrhosis, pulmonary fibrosis, dermatosclerosis,glomerular fibrosis and the like.

The pharmaceutically acceptable salt in the present invention can beprepared according to a conventional method well-known in the art, e.g.,by reacting the compound of the present invention with an inorganic acidsuch as hydrochloric acid, hydrobromide, sulfuric acid, sodium hydrogensulfate, phosphoric acid or carbonic acid; an organic acid such asformic acid, acetic acid, oxalic acid, benzoic acid, citric acid,tartaric acid, gluconic acid, gestisic acid, fumaric acid, lactobionicacid, salicylic acid, or acetylsalicylic acid (aspirin); an alkali metalion such as sodium or potassium; or ammonium ion.

Further, the pharmaceutical composition of the present invention mayfurther comprise a conventionally atoxic, pharmaceutically acceptablecarrier, adjuvant and excipient in addition to the benzopyran derivativeor the pharmaceutically acceptable salt thereof, and be formulated intoa conventional formulation in a pharmaceutical field, e.g., aformulation for oral administration such as tablets, capsules, troches,liquids or emulsions, or a formulation for parental administration.Further, for treating a human patient, a typical daily dose of theinventive compound as an active ingredient may range from about 0.01 to1000 mg/day based on an adult patient having 70 kg of body weight, andcan be administered in a single dose or in divided doses. However, itshould be understood that the amount of the active ingredient actuallyadministered ought to be determined in light of various relevant factorsincluding the condition to be treated, the chosen route ofadministration, the age, sex and body weight of the individual patient,and the severity of the patient's symptom; and, therefore, the abovedose should not be intended to limit the scope of the invention in anyway.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing antagonistic activity on TGF-β receptor ofN-(2,7-di substituted-2-methyl-2H-chromen-6-yl)thiourea derivative.

FIG. 2 is a graph showing antagonistic activity on TGF-β receptor ofN-(2,7-di substituted-2-methyl-2H-chromen-6-yl)-N,N′-disubstitutedguanidine derivative.

FIG. 3 is a graph showing the effect of N-(2,7-disubstituted-2-methyl-2H-chromen-6-yl)thiourea derivative on cytotoxicityand collagen synthesis in LI 90 cells.

FIG. 4 is a graph showing the effect of N-(2,7-disubstituted-2-methyl-2H-chromen-6-yl)-N,N′-disubstituted guanidinederivative on cytotoxicity and collagen synthesis in LI 90 cells.

FIG. 5 is a graph showing the effect of N-(2,7-disubstituted-2-methyl-2H-chromen-6-yl)thiourea derivative on theexpression of a collagen encoding gene in LI 90 cells.

FIG. 6 is a graph showing the effect of N-(2,7-disubstituted-2-methyl-2H-chromen-6-yl)-N,N′-disubstituted guanidinederivative on the expression of a collagen encoding gene in LI 90 cells.

FIG. 7 is a result of an animal experiment (in vivo) showing the effectof N-(2,7-di substituted-2-methyl-2H-chromen-6-yl)thiourea derivative onthe inhibition and treatment of hepatic fibrosis/liver cirrhosis.

The present invention is further illustrated in the following Examples.It should be understood that these Examples, while indicating preferredembodiments of the invention, are given by way of illustration only.

BEST MODE Example 1 Synthesis of Thiourea-Based Benzopyran DerivativeFormula 1a Example 1-1 Synthesis of1-(2,2′-dimethyl-2H-chromen-6-yl)-3-phenyl-thiourea

After a benzopyrene compound (100.0 mg, 0.57 mmol) was dissolved indichloromethane (DCM, 2 mL) and stirred at room temperature for 10 min,phenyl isothiocyanate of Formula (3a) (C₆H₄NCS; 92.0 mg, 0.68 mmol, 1.2eq) was added thereto, and the mixture was stirred at the sametemperature for 15 hrs. After the reaction was completed, polystyrenediamine of Formula (4) (3.0 mmol/g, 0.34 g, 1 mmol) was added to thereactant and stirred for 30 min. The reactant was subjected tofiltration to separate a filtrate. The filtrate was repeatedly washedwith chloroform (CHCl₃) and collected. The resulting reactant wasconcentrated under reduced pressure and the residue thus obtained waspurified with a silica gel column chromatography using a mixed solventof hexane/ethylacetate (4/1, v/v), to obtain the title compound (115 mg)with a yield of 65%.

¹H NMR (300 MHz, CDCl₃) δ 8.23 (d, 1H, J=7.9 Hz), 7.77 (s, 1H), 7.39 (s,1H), 7.38-7.27 (m, 2H), 7.14-7.08 (m, 3H), 7.00 (d, 2H), 6.82 (d, 1H,J=8.5 Hz), 6.30 (d, 1H, J=9.9 Hz), 5.69 (d, 1H, J=9.9 Hz), 1.45 (s, 6H);m/z 310.42

Example 1-2 Synthesis of1-(2,2′-dimethyl-2H-chromen-6-yl)-3-(4-nitrophenyl)-thiourea

After a benzopyrene compound (100.0 mg, 0.57 mmol) was dissolved indichloromethane (DCM, 15 mL) and stirred at room temperature for 10 min,4-nitrophenyl isothiocyanate of Formula (3b) (4-O₂NC₆H₄NCS; 122 mg, 0.68mmol, 1.2 eq) was added thereto, and the mixture was stirred at the sametemperature for 15 hrs. After the reaction was completed, polystyrenediamine of Formula (4) (3.0 mmol/g, 0.34 g, 1 mmol) was added to thereactant and stirred for 30 min. The reactant was subjected tofiltration to separate a filtrate. The filtrate was repeatedly washedwith chloroform (CHCl₃) and collected. The resulting reactant wasconcentrated under reduced pressure and the residue thus obtained waspurified with a silica gel column chromatography using a mixed solventof hexane/ethylacetate (4/1, v/v), to obtain the title compound (150 mg)with a yield of 74%.

¹H NMR (200 MHz, CDCl₃) δ 8.20 (d, 2H, J=9.2 Hz), 7.75 (d, 2H, J=9.2Hz), 7.07 (m, 1H), 6.94-6.83 (m, 2H), 6.30 (d, 1H, J=9.8 Hz), 5.72 (d,1H, F=9.8 Hz), 1.47 (s, 6H); m/z 355.44

Example 1-3 Synthesis of1-(2,7-dimethyl-2-propyl-2H-chromen-6-yl)-3-(4-nitrophenyl)-thiourea

The title compound was obtained according to the same method asdescribed in Example 1-1.

m/z 397.49

Example 1-4 Synthesis of1-(2-methyl-2-phenethyl-2H-chromen-6-yl)-3-(4-nitrophenyl)-thiourea

The title compound was obtained according to the same method asdescribed in Example 1-1.

m/z 445.54

Example 1-5 Synthesis of1-(2,7-dimethyl-2-phenethyl-2H-chromen-6-yl)-3-(4-nitrophenyl)-thiourea

The title compound was obtained according to the same method asdescribed in Example 1-1.

m/z 459.59

Further, the thiourea-based benzopyran derivative of Formula (1a), as atarget compound of the present invention, was synthesized according tothe same method as described in Example 1, and the results are shown inthe following Tables 1a to 1g.

TABLE 1a (1a)

Com. Structural No. R¹ R² R³ data 1-1 Ph H H ¹H NMR(300 MHz, CDCl₃): δ8.23(d, 1 H, J = 7.9 Hz), 7.77(s, 1 H), 7.39(s, 1 H), 7.38-7.27(m, 2 H),7.14-7.08(m, 3 H), 7.00(d, 2 H), 6.82(d, 1 H, J = 8.5 Hz), 6.30(d, 1 H,J = 9.9 Hz), 5.69(d, 1 H, J = 9.9 Hz), 1.45(s, 6 H); m/z 310.42 1-24-O₂N-Ph H H ¹H NMR(200 MHz, CDCl₃): δ 8.20(d, 2 H, J = 9.2 Hz), 7.75(d,2 H, J = 9.2 Hz), 7.07(m, 1 H), 6.94-6.83(m, 2 H), 6.30(d, 1 H, J = 9.8Hz), 5.72(d, 1 H, J = 9.8 Hz), 1.47(s, 6 H); m/z 355.44 1-3 3,4-di-Cl-PhH H ¹H NMR(300 MHz, CDCl₃): δ 7.73(br, 1 H), 7.59-7.58(m, 1 H),7.43-7.40(m, 1 H), 7.36-7.32(m, 1 H), 7.06-7.02(m, 1 H), 6.93-6.92(m, 1H), 6.85-6.82(m, 1 H), 6.28(d, 1 H, J = 9.9 Hz), 5.69(d, 1 H, J = 9.9 H)1-4 2,4-di-F-Ph H H ¹H NMR(300 MHz, CDCl₃): δ 8.26(br, 1 H),7.85-7.80(m, 1 H), 7.38(br, 1 H), 7.08-7.05(m, 1 H), 6.96-6.95(m, 1 H),6.93-6.81(m, 1 H), 6.28(d, 1 H, J = 9.9 Hz), 5.68(d, 1 H, J = 9.9 Hz),1.45 (s, 6 H) 1-5 adamantyl H H ¹H NMR(300 MHz, CDCl₃): δ 7.26-7.23(m, 1H), 6.94-6.91(m, 1 H), 6.81-6.76(m, 1 H), 6.28(d, 1 H, J = 9.9 Hz),5.70(d, 1 H, J = 9.9 Hz), 2.18(br, 6 H), 2.09(br, 2 H), 1.67(br, 6 H),1.60(s, 1 H), 1.44(s, 6 H) 1-6 2-ethyl-Ph H H ¹H NMR(300 MHz, CDCl₃): δ7.65(br, 1 H), 7.41-7.38(m, 1 H), 7.30-7.23(m, 4 H), 7.08-7.04(m, 1 H),6.99-6.98(m, 1 H), 6.78-6.76(m, 1 H), 6.26(d, 1 H, J = 9.8 Hz), 5.63(d,1 H, J = 9.8 Hz), 2.64(q, 2 H), 1.42(s, 6 H), 1.19(t, 3 H)

TABLE 1b 1-7 2,2,4- H H ¹H NMR(300 MHz, CDCl₃): δ 7.22(br, 1 H),Trimethyl- 6.94-6.90(m, 1 H), 6.80-6.78(m, 2 H), 6.27(d, 1 H, pentyl J =9.9 Hz), 5.84(br, 1 H), 5.68(d, 1 H, J = 9.9 Hz), 1.54(s, 6 H), 1.44(s,1 H), 0.91(s, 11 H) 1-8

H H ¹H NMR(300 MHz, CDCl₃): δ 7.59(br, 2 H), 7.43 7.29(m, 7 H),7.07-6.97(m, 4 H), 6.78(d, 1 H, J = 8.4 Hz), 6.27(d, 1 H, J = 9.9 Hz),5.64(d, 1 H, J = 9.9 Hz), 5.08(s, 2 H), 1.43(s, 6 H) 1-9 Phenethyl H H¹H NMR(300 MHz, CDCl₃): δ 7.74(br, 1 H), 7.27-7.20(m, 3 H), 7.14-7.11(m,2 H), 6.74-6.63(m, 3 H), 6.17(d, 1 H, J = 9.9 Hz), 5.81(br, 1 H),5.66(d, 1 H, J = 9.9 Hz), 3.86(t, 2 H), 2.90(t, 2 H), 1.43(s, 6 H) 1-10benzoyl H H ¹H NMR(300 MHz, CDCl₃): δ 12.40(br, 1 H), 9.08(br, 1 H),7.88(d, 2 H, J = 7.5 Hz), 7.87-7.51(m, 3 H), 7.38-7.35(m, 2 H), 6.80(d,1 H, J = 9.0 Hz), 6.32(d, 1 H, J = 9.9 Hz), 5.65(d, 1 H, J = 9.9 Hz),1.44(s, 6 H) 1-11 cyclohexyl H H ¹H NMR(200 MHz, CDCl₃): δ 7.61(br, 1H), 6.90(m, 1 H), 6.79-6.74(m, 2 H), 6.25(d, 1 H, J = 10.0 Hz), 5.67 (d,1 H, J = 10.0 Hz), 4.27-4.15(m, 1 H), 2.01(br, 2 H), 1.62(br, 3 H),1.43(s, 6 H), 1.35(br, 2 H), 1.09(br, 3 H) 1-12 2,5-di- H H ¹H NMR(200MHz, CDCl₃): δ 8.19(br, 2 H), 7.79(br, MeO-Ph 1 H), 7.09-7.03(m, 1 H),6.98-6.97(m, 1 H), 6.84-6.75(m, 2 H), 6.67-6.61(m, 1 H), 6.25(d, 1 H, J= 10.0 Hz), 5.67(d, 1 H, J = 10.0 Hz), 3.79(s, 3 H), 3.69(s, 3 H),1.45(s, 6 H) 1-13 1-naphthyl H H ¹H NMR(300 MHz, CDCl₃): δ 7.98(br, 1H), 7.93-7.86(m, 3 H), 7.60-7.50(m, 4 H), 7.07-6.97(m, 2 H), 6.75(d, 1H, J = 8.4 Hz), 6.26(d, 1 H, J = 9.6 Hz), 5.63(d, 1 H, J = 9.6 Hz),1.41(s, 6 H) 1-14 3-Cl-4- H H ¹H NMR(200 MHz, CDCl₃): δ 7.89(br, 1 H),7.59(br, Me-Ph 1 H), 7.39(s, 1 H), 7.22(s, 2 H), 7.07-7.02(m, 1 H),6.95-6.94(m, 1 H), 6.82-6.78(m, 1 H), 6.28(d, 1 H, J = 9.8 Hz), 5.67(d,1 H, J = 9.8 Hz), 2.34(s, 3 H), 1.44(s, 6 H) 1-15

H H ¹H NMR(300 MHz, CDCl₃): δ 7.85(br, 1 H), 7.67(br, 1 H), 7.30(d, 2 H,J = 8.61 Hz), 7.30(d, 2 H, J = 8.61 Hz), 7.25-7.23(d, 2 H, J = 8.61 Hz),7.07 7.04(m, 1 H), 6.96-6.95(m, 1 H), 6.79(d, 1 H, J = 8.4 Hz), 6.27(d,1 H, J = 9.9 Hz), 5.65(d, 1 H, J = 9.9 Hz), 2.47(s, 3 H), 1.44(s, 6 H)

TABLE 1c 1-16

H H ¹H NMR(200 MHz, CDCl₃): δ 7.79(br, 1 H), 7.66(br, 1 H), 7.31(s, 1H), 7.30-7.23(m, 1 H), 7.15-7.02(m, 3 H), 6.96-6.95(m, 1 H), 6.78(d, 1H, J = 8.5 Hz), 6.27(d, 1 H, J = 10.0 Hz), 5.66(d, 1 H, J = 10.0 Hz),2.47(s, 3 H), 1.44 (s, 6 H) 1-17 2-isopropyl- H H ¹H NMR(300 MHz,CDCl₃): δ 7.65(br, 1 H), 7.38 7.22(m, 4 H), Ph 7.07-6.98(m, 1 H),6.76(d, 1 H, J = 8.5 Hz), 6.26(d, 1 H, J = 9.8 Hz), 5.63(d, 1 H, J = 9.8Hz), 3.17-3.13(m, 1 H), 1.42(s, 6 H), 1.24(s, 6 H) 1-18 5-Cl-2-Me H H ¹HNMR(200 MHz, CDCl₃): δ 7.09-6.95(m, 3 H), 6.85-6.73(m, O-Ph 3 H),6.31(d, 1 H, J = 9.8 Hz), 5.69(m, 1 H, J = 9.8 Hz), 3.73(s, 3 H),1.46(s, 6 H) 1-19

H H ¹H NMR(300 MHz, CDCl₃): δ 8.41(br, 1 H), 7.98(s, 2 H), 7.67(s, 2 H),7.07-7.03(m, 1 H), 6.93-6.92(m, 1 H), 6.84(d, 1 H, J = 8.5 Hz), 6.28(d,1 H, J = 9.9 Hz), 5.71(d, 1 H, J = 9.9 Hz), 1.46(s, 6 H) 1-20

H H ¹H NMR(200 MHz, CDCl₃): δ 8.02(br, 1 H), 7.28-7.24(m, 3 H),6.89-6.67(m, 3 H), 6.23(d, 1 H, J = 9.8 Hz), 5.58(d, 1 H, J = 9.8 Hz),3.36-3.22(m, 2 H), 1.44-1.09(m, 18 H) 1-21 3,5-di- H H ¹H NMR(200 MHz,CDCl₃): δ 7.82(br, 2 H), 7.08 6.89(m, 5 H), Me-Ph 6.75(d, 1 H, J = 8.5Hz), 6.27(d, 1 H, J = 9.6 Hz), 5.64(d, 1 H, J = 9.6 Hz), 2.30(s, 6 H),1.42(s, 6 H) 1-22

H H ¹H NMR(200 MHz, CDCl₃): δ 8.30(br, 1 H), 7.89(d, 1 H, J = 8.5 Hz),7.73-7.64(m, 2 H), 7.48(br, 1 H), 7.07-7.02(m, 1 H), 6.95-6.93(m, 1 H),6.82(d, 1 H, J = 8.3 Hz), 6.29(d, 1 H, J = 9.8 Hz), 5.68(d, 1 H, J = 9.8Hz), 1.43(s, 6 H) 1-23 2-F-Ph H H ¹H NMR(200 MHz, CDCl₃): δ 8.06-8.01(m,1 H), 7.98(br, 1 H), 7.53(br, 1 H), 7.22-7.04(m, 4 H), 6.98-6.96(m, 1H), 6.82(d, 1 H, J = 8.5 Hz), 6.29(d, 1 H, J = 9.8 Hz), 5.68(d, 1 H, J =9.8 Hz), 1.45(s, 6 H) 1-24 3-Me-Ph H H ¹H NMR(200 MHz, CDCl₃): δ7.80(br, 2 H), 7.27-7.16(m, 4 H), 7.09-7.04(m, 1 H), 6.98-6.97(m, 1 H),6.77(d, 1 H, J = 8.3 Hz), 6.28(d, 1 H, J = 9.8 Hz), 5.65(d, 1 H, J = 9.8Hz), 2.35(s, 3 H), 1.43(s, 6 H) 1-25 3-MeO-Ph H H ¹H NMR(300 MHz,CDCl₃): δ 7.58(br, 2 H), 7.30-7.24(m, 2 H), 7.09-6.89(m, 4 H), 6.78(d, 1H, J = 8.5 Hz), 6.28(d, 1 H, J = 9.8 Hz), 5.65(d, 1 H, J = 9.8 Hz),3.81(s, 3 H), 1.44(s, 6 H)

TABLE 1d 1-26 Cyclopentyl H H ¹H NMR(200 MHz, CDCl₃): δ 7.62(br, 1 H),6.95 .81(m, 1 H), 6.80-6.76(m, 2 H), 6.27(d, 1 H, J = 10.0 Hz), 5.67(d,1 H, J = 10.0 Hz), 4.70-4.59(m, 1 H), 2.12-1.99(br, 2 H), 1.67-1.55(br,4 H), 1.44(s, 1 H), 1.40 1.25(br, 2 H) 1-27

H H ¹H NMR(300 MHz, CDCl₃): δ 7.84(br, 1 H), 7.27 7.23(m, 2 H),7.03-6.89(m, 3 H), 6.78-6.75(m, 2 H), 6.23(d, 1 H, J = 9.6 Hz), 6.01(br,1 H), 5.66(d, 1 H, J = 9.6 Hz), 1.44(s, 12 H) 1-28 Undecyl H H ¹HNMR(200 MHz, CDCl₃): δ 7.66(br, 1 H), 6.97-6.91(m, 1 H), 6.83-6.77(m, 2H), 6.26(d, 1 H, J = 9.8 Hz), 5.68(d, 1 H, J = 9.8 Hz), 3.64-3.54(m, 2H), 1.45(s, 6 H), 1.25(s, 9 H), 0.88(t, 3 H) 1-29

H H ¹H NMR(200 MHz, CDCl₃): δ 8.03(br, 1 H), 8.03(br, 1 H), 7.73-7.68(m,1 H), 7.52-7.45(m, 2 H), 7.09-7.03(m, 1 H), 6.94-6.93(m, 1 H), 6.85(d, 1H, J = 8.5 Hz), 6.30(d, 1 H, J = 10.0 Hz), 5.71(d, 1 H, J = 10.0 Hz),1.47(s, 6 H) 1-30 Heptyl H H ¹H NMR(200 MHz, CDCl₃): δ 7.69(br, 1 H),6.97-6.91(m, 1 H), 6.83-6.77(m, 2 H), 6.26(d, 1 H, J = 10.0 Hz),5.82(br, 1 H), 5.68(d, 1 H, J = 10.0 Hz), 3.64-3.54(m, 2 H), 1.45(s, 6H), 1.26(s, 9 H), 0.87(t, 3 H) 1-31

H H ¹H NMR(200 MHz, CDCl₃): δ 8.31(br, 1 H), 7.72-7.65(m, 2 H),7.47-7.44(m, 2 H), 7.08-7.02(m, 1 H), 6.94-6.93(m, 1 H), 6.81(d, 1 H, J= 8.5 Hz), 6.28(d, 1 H, J = 9.8 Hz), 1.45(d, 1 H, J = 9.8 Hz), 1.45(s, 6H) 1-32 2-MeO-Ph H H ¹H NMR(200 MHz, CDCl₃): δ 8.02(br, 1 H), 7.83(br, 1H), 7.18-6.94(m, 4 H), 6.89-6.79(m, 3 H), 6.29(d, 1 H, J = 9.8 Hz),5.67(d, 1 H, J = 9.8 Hz), 3.76(s, 3 H), 1.45(s, 6 H) 1-33

H H ¹H NMR(200 MHz, CDCl₃): δ 7.93(br, 1 H), 7.46(s, 1 H), 7.17-7.13(m,2 H), 7.9-7.03(m, 1 H), 6.97-6.95(m, 1 H), 6.79(d, 1 H, J = 8.5 Hz),6.28(d, 1 H, J = 10.0 Hz), 5.67(d, 1 H, J = 10.0 Hz), 2.22(s, 3 H),1.44(s, 6 H) 1-34 4-MeO-Ph H H ¹H NMR(300 MHz, CDCl₃): δ 7.69(br, 2 H),7.27(d, 2 H, J = 9.0 Hz), 7.08-7.04(m, 1 H), 6.98-6.97(m, 1 H), 6.91(d,2 H, J = 9.0 Hz), 6.77(d, 1 H, J = 8.4 Hz), 6.27(d, 1 H, J = 9.9 Hz),5.63(d, 1 H, J = 9.9 Hz), 3.80(s, 3 H), 1.43(s, 6 H)

TABLE 1e 1-35 Octyl H H ¹H NMR(300 MHz, CDCl₃): δ 7.70(br, 1 H),6.97-6.91(m, 1 H), 6.83-6.77(m, 2 H), 6.26(d, 1 H, J = 9.8 Hz), 5.83(br,1 H), 5.68(d, 1 H, J = 9.8 Hz), 3.64-3.54(m, 2 H), 1.71 1.51(m, 2 H),1.45(s, 6 H), 1.25(s, 10 H), 0.87(t, 3 H) 1-36

H H ¹H NMR(300 MHz, CDCl₃): δ 7.84(br, 1 H), 7.27-7.23(m, 2 H),7.03-6.89(m, 3 H), 6.78-6.75(m, 2 H), 6.23(d, 1 H, J = 9.6 Hz), 6.01(br,1 H), 5.66(d, 1 H, J = 9.6 Hz), 1.44(s, 12 H) 1-37 2-Cl-Ph H H ¹HNMR(300 MHz, CDCl₃): δ 7.93(br, 1 H), 7.83(br, 1 H), 7.39-7.27(m, 4 H),7.08-7.04(m, 1 H), 6.98-6.97(m, 1 H), 6.77(d, 1 H, J = 8.5 Hz), 6.27(d,1 H, J = 9.8 Hz), 5.65(d, 1 H, J = 9.8 Hz), 1.44(s, 6 H) 1-382,6-di-Me-Ph H H ¹H NMR(300 MHz, CDCl₃): δ 7.17-6.99(m, 6 H), 6.27(d, 1H, J = 9.0 Hz), 5.61(d, 1 H, J = 9.0 Hz), 2.35(s, 6 H), 1.43(s, 6 H)1-39 3,5-di-methoxy- H H ¹H NMR(200 MHz, CDCl₃): δ 6.78(br, 1 H),6.70(s, Ph 1 H), 6.58(s, 1 H), 6.23(d, 1 H, J = 10.0 Hz), 5.49(d, 1 H, J= 10.0 Hz), 3.75-3.73(br, 4 H), 2.15(s, 3 H), 1.69-1.56(br, 6 H),1.52-1.36(m, 2 H), 1.34(s, 6 H), 0.89(t, 3 H, J = 7.1 Hz) 1-403,5-di-Me-Ph Me H ¹H NMR(200 MHz, CDCl₃): δ 7.54(br, 1 H), 6.98-6.88(m,4 H), 6.69(s, 1 H), 6.25(d, 1 H, J = 10.0 Hz), 5.59(d, 1 H, J = 10.0Hz), 2.31(s, 6 H), 2.30(s, 3 H), 1.43(s, 6 H) 1-41 Cyclopentyl Me H ¹HNMR(200 MHz, CDCl₃): δ 7.31(br, 1 H), 6.78(s, 1 H), 6.69(s, 1 H),6.25(d, 1 H, J = 9.8 Hz), 5.63(d, 1 H, J = 9.8 Hz), 5.48-5.45(m, 1 H),4.71-4.61(m, 1 H), 2.17(s, 3 H), 2.14-1.99(br, 2 H), 1.61-1.49(br, 4 H),1.44(s, 6 H), 1.39-1.25(br, 2 H) 1-42 heptyl Me H ¹H NMR(200 MHz,CDCl₃): δ 7.40(br, 1 H), 6.79(s, 1 H), 6.70(s, 1 H), 6.25(d, 1 H, J =10.0 Hz), 5.63(d, 1 H, J = 10.0 Hz), 3.63-3.53(m, 2 H), 2.18(s, 3 H),1.71 1.48(m, 2 H), 1.44(s, 6 H), 1.24(s, 8 H), 0.87(t, 3 H) 1-43

Me H ¹H NMR(200 MHz, CDCl₃): δ 7.93(br, 1 H), 6.94(s, 1 H), 6.75(s, 1H), 6.54(br, 1 H), 6.28(d, 1 H, J = 9.8 Hz), 5.65(d, 1 H, J = 9.8 Hz),2.30(s, 3 H), 1.45(s, 6 H) 1-44 benzoyl Me H ¹H NMR(200 MHz, CDCl₃): δ9.20(br, 1 H), 7.93 7.88(m, 2 H), 7.66-7.50(m, 3 H), 7.32(s, 1 H),6.70(s, 1 H), 6.30(d, 1 H, J = 9.8 Hz), 5.59(d, 1 H, J = 9.8 Hz),2.28(s, 3 H), 1.44(s, 6 H)

TABLE 1f 1-45 undecyl Me H ¹H NMR(200 MHz, CDCl₃): δ 7.25(br, 1 H),6.78(s, 1 H), 6.70(s, 1 H), 6.24(d, 1 H, J = 9.8 Hz), 5.62(d, 1 H, J =9.8 Hz), 3.63-3.53(m, 2 H), 2.17(s, 3 H), 1.43(s, 6 H), 1.23(s, 8 H),0.87(t, 3 H) 1-46 2-Cl-Ph Me H ¹H NMR(200 MHz, CDCl₃): δ 8.17(br, 1 H),7.98-7.97(m, 2 H), 7.66(s, 1 H), 7.42(br, 1 H), 6.90(s, 1 H), 6.75(s, 1H), 6.28(d, 1 H, J = 9.8 Hz), 5.65(d, 1 H, J = 9.8 Hz), 2.28(s, 3 H),1.45(s, 6 H) 1-47 2-Cl-Ph Me H m/z 358.9 1-48 3-Cl-4-Me-Ph Me H m/z372.9 1-49 2,6-di-Me-Ph Me H m/z 352.5 1-50 2-ethyl-Ph Me H m/z 352.51-51 3-methyl Me H m/z 370.5 sulfanyl-Ph 1-52 2-MeO-Ph Me H m/z 354.51-53 3-Cl-2-Me-Ph Me H ¹H NMR(200 MHz, CDCl₃): δ 7.65(br, 1 H),7.20-7.11(m, 2 H), 6.91(s, 1 H), 6.71(s, 1 H), 6.25(d, 1 H), J = 9.8Hz), 5.62(d, 1 H, J = 9.8 Hz), 2.27(s, 3 H), 2.18(d, 3 H), 1.42(s, 6 H)1-54 4-NO₂-Ph Me H m/z 369.4 1-55 3-trifluoro Me H m/z 392.4 1-56phenethyl Me H m/z 352.5 1-57 4-MeO-Ph Me CH₂CH₃ m/z 366.5 1-58 4-Me-PhMe CH₂CH₃ m/z 350.5 1-59 4-Cl-Ph Me CH₂CH₃ m/z 370.9 1-60 4-O₂N-Ph MeCH₂CH₃ m/z 381.4 1-61 4-F-Ph Me CH₂CH₃ m/z 354.4 1-62 3,4-di-Me-Ph MeCH₂CH₃ m/z 364.5 1-63

Me CH₂CH₃ ¹H NMR(200 MHz, CDCl₃): δ 7.31-7.21(m, 2 H), 6.92-6.85(m, 4H), 6.73(s, 1 H), 6.61(s, 1 H), 6.24(d, 1 H, J = 10.0 Hz), 5.49(d, 1 H,J = 10.0 Hz), 3.98-3.93(m, 4 H), 3.27-3.21(m, 4 H), 2.17(s, 3 H),1.71-1.57(m, 2 H), 1.51-1.38(m, 2 H), 1.35(s, 3 H), 0.93-0.86(m, 3 H)1-64 4-Cl-Bn Me CH₂CH₃ m/z 400.9 1-65

Me CH₂CH₃ ¹H NMR(200 MHz, CDCl₃): δ 6.87(br, 1 H), 6.68(s, 1 H), 6.58(s,1 H), 6.23(d, 1 H, J = 10.2 Hz), 5.48(d, 1 H, J = 10.2 Hz), 4.35(br,2H), 3.67(s, 3 H), 3.22(br, 2 H), 2.59-2.49(m, 1 H), 2.14(s, 3 H),2.02-1.50(br, 6 H), 1.46-1.34(m, 5 H), 0.88(t, 3 H, J = 7.3 Hz)

TABLE 1g 1-66

Me CH₂CH₃ ¹H NMR(200 MHz, CDCl₃): δ 6.78(br, 1 H), 6.70(s, 1 H), 6.58(s,1 H), 6.23(d, 1 H, J = 10.0 Hz), 5.49(d, 1 H, J = 10.0 Hz),3.75-3.73(br, 4 H), 2.15(s, 3 H), 1.69-1.56(br, 6 H), 1.52-1.36(m, 2 H),1.34(s, 6 H), 0.89(t, 3 H, J = 7.1 Hz) 1-67

Me CH₂CH₃ ¹H NMR(200 MHz, CDCl₃): δ 9.08(br, 1 H), 7.00(s, 1 H), 6.61(s,1 H), 6.28(d, 1 H, J = 9.8 Hz), 5.47(d, 1 H, J = 9.8 Hz), 3.71(s, 3 H),2.17(s, 3 H), 1.74-1.57(m, 2 H), 1.51-1.38(m, 2 H), 1.35(s, 3 H),0.89(t, 3 H, J = 7.1 Hz) 1-68 4-MeO-Ph H CH₂Ph m/z 414.5 1-69 4-Me-Ph HCH₂Ph m/z 398.5 1-70 4-Cl-Ph H CH₂Ph m/z 418.9 1-71 4-O₂N-Ph H CH₂Ph m/z429.5 1-72 4-F-Me-Ph H CH₂Ph m/z 402.5 1-73 3,4-di-Me-Ph H CH₂Ph m/z412.5 1-74 4-MeO-Ph Me CH₂Ph m/z 428.5 1-75 4-Me-Ph Me CH₂Ph m/z 412.51-76 4-Cl-Ph Me CH₂Ph m/z 432.9 1-77 4-O₂N-Ph Me CH₂Ph m/z 443.5 1-784-F-Me-Ph Me CH₂Ph m/z 416.5 1-79 3,4-di-Me-Ph Me CH₂Ph m/z 426.6

Example 2 Synthesis of Guanidine-Based Benzopyran Derivative Formula 1bExample 2-1 Synthesis ofN-(2,2′-dimethyl-2-H-chromen-6-yl)-N′-(4-nitrophenyl)-N″-phenyl-guanidine

A thiourea compound (50 mg, 0.16 mmol, 1 eq) was added to chloroform(CHCl₃, 5 mL) and stirred at room temperature for 10 min.1,3-Diisopropylcarbodiimide (DIC; 0.029 mL, 0.19 mmol, 1.2 eq) anddiisopropylethylamine (DIPEA; 0.033 mL, 0.19 mmol, 1.2 eq) were addedthereto and stirred at 50° C. for 10 min. Subsequently, 4-nitroanilineof Formula (5a) (44 mg, 0.32 mmol) was added thereto and stirred for 15hrs. After the reaction was completed, the resulting mixture was cooleddown to room temperature, polystyrene isocyanate of Formula (6) (2.90mmol/g, 0.35 g, 1 mmol) was added thereto, and stirred for 30 min. Afterthe reaction mixture was filtered, the filtrate thus obtained wasrepeatedly washed with chloroform (CHCl₃) and collected. The resultingreactant was concentrated under reduced pressure and the residue thusobtained was purified with a silica gel column chromatography using amixed solvent of hexane/ethylacetate (3/1, v/v), to obtain the titlecompound (35 mg) with a yield of 53%.

Example 2-2 Synthesis ofN-(2,2′-dimethyl-2H-chromen-6-yl)-N′-(4-nitrophenyl)-N″-4-tolyl-guanidine

A thiourea compound (69 mg, 0.16 mmol, 1 eq) was added to chloroform(CHCl₃, 5 mL) and stirred at room temperature for 10 min.1,3-Diisopropylcarbodiimide (DIC; 0.029 mL, 0.19 mmol, 1.2 eq) anddiisopropylethylamine (DIPEA; 0.033 mL, 0.19 mmol, 1.2 eq) were addedthereto and stirred at 50° C. for 10 min. Subsequently, 4-methylanilineof Formula (5b) (0.035 mL, 0.32 mmol) was added thereto and stirred for15 hrs. After the reaction was completed, the resulting mixture wascooled down to room temperature, polystyrene isocyanate of Formula (6)(2.90 mmol/g, 0.35 g, 1 mmol) was added thereto, and stirred for 30 min.After the reaction mixture was filtered, the filtrate thus obtained wasrepeatedly washed with chloroform (CHCl₃) and collected. The resultingreactant was concentrated under reduced pressure and the residue thusobtained was purified with a silica gel column chromatography using amixed solvent of hexane/ethylacetate (3/1, v/v), to obtain the titlecompound (34 mg) with a yield of 49%.

Example 2-3 Synthesis ofN-(2,2′-dimethyl-2H-chromen-6-yl)-N′-(4-fluorophenyl)-N″-(4-nitrophenyl)-guanidine

A thiourea compound (64 mg, 0.16 mmol, 1 eq) was added to chloroform(CHCl₃, 5 mL) and stirred at room temperature for 10 min.1,3-Diisopropylcarbodiimide (DIC; 0.029 mL, 0.19 mmol, 1.2 eq) anddiisopropylethylamine (DIPEA; 0.033 mL, 0.19 mmol, 1.2 eq) were addedthereto and stirred at 50° C. for 10 min. Subsequently, 4-fluoroanilineof Formula (5c) (0.030 mL, 0.32 mmol) was added thereto and stirred for15 hrs. After the reaction was completed, the resulting mixture wascooled down to room temperature, polystyrene isocyanate of Formula (6)(2.90 mmol/g, 0.35 g, 1 mmol) was added thereto, and stirred for 30 min.After the reaction mixture was filtered, the filtrate thus obtained wasrepeatedly washed with chloroform (CHCl₃) and collected. The resultingreactant was concentrated under reduced pressure and the residue thusobtained was purified with a silica gel column chromatography using amixed solvent of hexane/ethylacetate (3/1, v/v), to obtain the titlecompound (44 mg) with a yield of 58%.

Example 2-4 Synthesis ofN-(2,2′-dimethyl-2H-chromen-6-yl)-N′-(4-methoxyphenyl)-N″-(4-nitrophenyl)-guanidine

A thiourea compound (71 mg, 0.16 mmol, 1 eq) was added to chloroform(CHCl₃, 5 mL) and stirred at room temperature for 10 min.1,3-Diisopropylcarbodiimide (DIC; 0.029 mL, 0.19 mmol, 1.2 eq) anddiisopropylethylamine (DIPEA; 0.033 mL, 0.19 mmol, 1.2 eq) were addedthereto and stirred at 50° C. for 10 min. Subsequently, 4-methoxyanilineof Formula (5d) (39 mg, 0.32 mmol) was added thereto and stirred for 15hrs. After the reaction was completed, the resulting mixture was cooleddown to room temperature, polystyrene isocyanate of Formula (6) (2.90mmol/g, 0.35 g, 1 mmol) was added thereto, and stirred for 30 min. Afterthe reaction mixture was filtered, the filtrate thus obtained wasrepeatedly washed with chloroform (CHCl₃) and collected. The resultingreactant was concentrated under reduced pressure and the residue thusobtained was purified with a silica gel column chromatography using amixed solvent of hexane/ethylacetate (3/1, v/v), to obtain the titlecompound (37 mg) with a yield of 43%.

The guanidine-based benzopyran derivative of Formula (1b), as a targetcompound of the present invention was synthesized according to the samemethod as described in Example 2, and the results are shown in thefollowing Tables 2a to 2e.

TABLE 2a (1b)

Com. Structural No R¹ R² R³ R⁴ data 2-1 Ph H H Ph m/z 369.48 2-2 Ph H H4-Me-Ph- m/z 383.49 2-3 Ph H H 4-F-Ph- ¹H NMR(300 MHz, CDCl₃) δ7.36-7.31(m, 5 H), 7.01(m, 2 H), 6.86(t, 2 H, J = 8.7 Hz), 6.65(d, 1 H),6.64-6.61(m, 2 H), 6.32(d, 1 H), 5.72(d, 1 H), 1.43(s, 6 H); m/z 387.462-4 Ph H H 4-Cl-Ph- m/z 403.92 2-5 Ph H H 4-MeO-Ph- ¹H NMR(300 MHz,CDCl₃) δ 7.29-7.11(m, 7 H), 6.95(d, 2 H, J = 8.1 Hz), 6.86-6.81(m, 2 H),6.67(d, 2 H, J = 7.2 Hz), 6.25(d, 1 H), 5.62(d, 1 H, J = 9.9 Hz),3.75(s, 3 H), 1.38(s, 6 H); m/z 399.47 2-6 Ph H H 4-NO₂-Ph- ¹H NMR(300MHz, CDCl₃) δ 8.08(d, 2 H, J = 8.9 Hz), 7.34-7.16(m, 7 H), 6.90(dd, 1 H,J = 8.6 Hz, J = 2.5 Hz), 6.82(d, 1 H, J = 2.5 Hz), 6.71(d, 1 H, J = 8.6Hz), 6.23(d, 1 H, J = 9.8 Hz), 5.65(d, 1 H, J = 9.8 Hz), 1.41(s, 6 H);m/z 414.46 2-7 Ph H H Bn— m/z 383.47 2-8 4-O₂N-Ph H H 4-Me-Ph- ¹HNMR(300 MHz, CDCl₃) δ 8.02(d, 2 H, J = 8.9 Hz), 7.21(d, 2 H, J = 8.9Hz), 7.08(s, 4 H), 6.1 (dd, 1 H, J = 8.4 Hz, J = 2.4 Hz), 6.84(d, 1 H, J= 2.4 Hz), 6.66(d, 1 H, J = 8.4 Hz), 6.21(d, 1 H, J = 9.9 Hz), 5.63(d, 1H, J = 9.9 Hz), 2.27(s, 3 H), 1.38(s, 6 H); m/z 428.50 2-9 4-O₂N-Ph H H3,5-Me-Ph- ¹H NMR(300 MHz, CDCl₃) δ 8.05(d, 2 H, J = 8.97 Hz), 7.20(d, 2H, J = 8.97 Hz), 6.89 (dd, 1 H, J = 8.46 Hz, J = 2.49 Hz), 6.82(d, 1 H,J = 2.49 Hz), 6.77(s, 3 H), 6.69(d, 1 H, J = 8.46 Hz), 6.22(d, 1 H, J =9.83 Hz), 5.64(d, 1 H, J = 9.83 Hz), 2.24(s, 6 H), 1.39(s, 6 H); m/z442.55

TABLE 2b 2-10 4-O₂N-Ph H H 4-F-Ph- ¹H NMR(300 MHz, CDCl₃) δ 8.10(d, 2H,J = 9.0 Hz), 7.26(d, 2H, J = 9.0 Hz), 7.17(m, 2H), 6.99(m, 2H), 6.91(dd, 1H, J = 8.4 Hz, J = 2.4 Hz), 6.81(d, 1H, J = 2.4 Hz), 6.73(d, 1H, J= 8.4 Hz), 6.24(d, 1H, J = 9.9 Hz), 5.66(d, 1H, J = 9.9 Hz), 1.42(s,6H); m/z 432.47 2-11 4-O₂N-Ph H H 4-Cl-Ph- ¹H NMR(300 MHz, CDCl₃) δ8.12(d, 2H, J = 7.6 Hz), 7.29-7.14(m, 4H), 7.10(d, 2H, J = 8.7 Hz),6.90(d, 1H, J = 2.6 Hz), 6.79-6.71(m, 2H), 6.23(d, 1H, J = 9.8 Hz),5.66(d, 1H, J = 9.8 Hz), 1.40(s, 6H); m/z 448.93 2-12 4-O₂N-Ph H H4-MeO-Ph- ¹H NMR(300 MHz, CDCl₃) δ 8.11(d, 2H, J = 8.9 Hz), 7.24(d, 2H,J = 8.9 Hz), 7.14(d, 2H, J = 8.8 Hz), 6.93(dd, 1H, J = 8.5 Hz, J = 2.3Hz), 6.86(m, 3H), 6.73(d, 1H, J = 8.5 Hz), 6.25(d, 1H, J = 9.8 Hz),5.64(d, 1H, J = 9.8 Hz), 3.78(s, 3H), 1.42(s, 6H); m/z 444.50 2-134-O₂N-Ph H H 2-MeO-Ph- m/z 444.51 2-14 4-O₂N-Ph H H iso-valine m/z452.54 (methyl ester)- 2-15 4-O₂N-Ph H H Bn- m/z 428.48 2-16 4-O₂N-Ph HH 4-F-Bn- ¹H NMR(300 MHz, CDCl₃) δ 8.14(d, 2H, J = 8.99 Hz), 7.27(m,2H), 7.12(d, 2H, J = 8.99 Hz), 7.03(m, 2H), 6.85(dd, 1H, J = 8.49 Hz, J= 2.60 Hz), 6.74(d, 1H, J = 2.60 Hz), 6.70(d, 1H, J = 8.49 Hz), 6.20(d,1H, J = 9.85 Hz), 5.65(d, 1H, J = 9.85 Hz), 4.45(s, 2H), 1.41(s, 6H);m/z Z 446.49 2-17 4-O₂N-Ph H H 4-Cl-Bn- m/z 462.95 2-18 4-O₂N-Ph H H4-NO₂-Ph- ¹H NMR(300 MHz, CDCl₃) δ 8.14(d, 2H, J = 9.32 Hz), 8.12(d, 2H,J = 8.49 Hz), 6.96(d, 2H, J = 8.49 Hz), 6.82(d, 2H, J = 9.32 Hz),6.70(m, 2H), 6.62(s, 1H), 6.22(d, 1H, J = 9.81 Hz), 5.66(d, 1H, J = 9.81Hz), 3.55(t, 4H, J = 4.11 Hz), 3.45(br-s, 4H), 1.41(s, 6H); m/z 528.552-19 4-O₂N-Ph H H Ph- ¹H NMR(300 MHz, CDCl₃) δ 8.10(d, 2H, J = 8.7piperazine- Hz), 7.30(d, 2H, J = 8.7 Hz), 6.92(m, 5H), 6.75(br-s, 1H),6.68(d, 1H, J = 8.1 Hz), 6.63(br-s, 1 H), 6.22(d, 1 H, J = 9.9 Hz),5.64(d, 1H, J = 9.9 Hz), 3.54(t, 4H, J = 5.0 Hz), 3.18(br-s, 4H),1.41(s, 6H); m/z 483.57

TABLE 2c 2-20 4-O₂N-Ph H H 2-MeO-Ph- ¹H NMR(300 MHz, CDCl₃) δ 8.10(d,2H, J = 8.7 Hz), piperazine- 7.06-6.92(m, 4H), 6.88(d, 2H, J = 8.7 Hz),6.74(br-s, 1H), 6.68(d, 1H, J = 8.1 Hz), 6.62(br-s, 1H), 6.22(d, 1H, J =9.9 Hz), 5.64(d, 1H, J = 9.9 Hz), 3.86(s, 3H), 3.57(t, 4H, J = 4.8 Hz),3.06(br-s, 4H), 1.41(s, 6H); m/z 513.63 2-21 4-O₂N-Ph H H 3-MeO-Ph- ¹HNMR(300 MHz, CDCl₃) δ 8.08(d, 2H, J = 8.40 piperazine- Hz), 7.18(m, 1H),6.93(d, 2H, J = 8.4 Hz), 6.75(br-s, 1H), 6.67(d, 1H, J = 8.40 Hz),6.63(br-s, 1H), 6.53(d, 1H, J = 9.03 Hz), 6.45(m, 2H), 6.21 (d, 1H, J =9.78 Hz), 5.64(d, 1H, J = 9.78 Hz), 3.79(s, 3H), 3.52(t, 4H, J = 4.91Hz), 3.17(br-s, 4H), 1.41(s, 6H); m/z 513.64 2-22 4-O₂N-Ph H H 4-F-Ph-¹H NMR(300 MHz, CDCl₃) δ 8.11(d, 2H, J = 9.0 Hz), piperazine-7.03-6.95(m, 3H), 6.91-6.85(m, 2H), 6.74(br-s, 1H), 6.69(d, 1H, J = 8.1Hz), 6.62(br-s, 1H), 6.22(d, 1H, J = 9.9 Hz), 5.65(d, 1H, J = 9.9 Hz),3.53(t, 4H, J = 5.0 Hz), 3.09(br-s, 4H), 1.41(s, 6H); m/z 501.58 2-234-O₂N-Ph H H 4-Cl-Ph- ¹H NMR(300 MHz, CDCl₃) δ 8. 10(d, 2H, J = 8.7 Hz),piperazine- 7.22(d, 2H, J = 7.5 Hz), 6.94(d, 2H, J = 8.7 Hz), 6.83(d,2H, J = 7.5 Hz), 6.75(br-s, 1H), 6.68(d, 1H, J = 8.4 Hz), 6.62(br-s,1H), 6.21 (d, 1H, J = 9.9 Hz), 5.64(d, 1H, J = 9.9 Hz), 3.52(t, 4H, J =5.3 Hz), 3.13(br-s, 4H), 1.41(s, 6H); m/z 518.03 2-24 4-O₂N-Ph H H4-Me-Ph- m/z 497.61 piperazine- 2-25 4-O₂N-Ph H H 3,4-dioxo- ¹H NMR(300MHz, CDCl₃) δ 8.14(d, 2H, J = 8.9 Hz), Bn- 7.16(d, 2H, J = 8.9 Hz),6.87(dd, 1H, J = 8.5 Hz, J = 2.5 Hz), 6.81-6.76(m, 4H), 6.70(d, 1H, J =8.5 Hz), 6.21 (d, 1H, J = 9.8 Hz), 5.96(s, 2H), 5.64(d, 1H, J = 9.8 Hz),4.42(s, 2H), 1.40(s, 6H); m/z 472.51 2-26 4-O₂N-Ph CH₃ —CH₂CH₃ Ph- ¹HNMR(300 MHz, CDCl₃) δ 8.13(d, 2H), 7.33-7.23(m, 7H), 7.10(t, 1H),6.82(s, 1H), 6.65(s, 1H), 6.27(d, 1H), 5.56(d, 1H), 5.64(d, 1H, J = 9.8Hz), 2.20(s, 3H), 1.62(m, 2H), 1.46(m, 2H), 1.37(s, 3H), 0.92(t, 3H);m/z 456.54

TABLE 2d 2-27 4-O₂N-Ph CH₃ —CH₂CH₃ 4-Me-Ph- ¹H NMR(300 MHz, CDCl₃) δ8.06(d, 2H), 7.22(d, 2H), 7.05(m, 4H), 6.82(s, 1H), 6.59(s, 1H), 6.25(d,1H), 5.54(d, 1H), 2.28(s, 3H), 2.04(s, 3H), 1.64(m, 2H), 1.58(m, 2H),1.37(s, 3H), 0.90(t, 3H); m/z 470.58 2-28 4-O₂N-Ph CH₃ —CH₂Cn3 4-MeO-Ph-¹H NMR(300 MHz, CDCl₃) δ 8.10(d, 2H), 7.28(d, 2H), 7.15(d, 2H), 6.86(m,3H), 6.62(s, 1H), 6.26(d, 1H), 5.55(d, 1H), 3.78(s, 3H), 2.20(s, 3H),1.66(m, 2H), 1.60(m, 2H), 1.40(s, 3H), 0.91 (t, 3H); m/z 486.56 2-294-O₂N-Ph CH₃ —CH₂CH₃ 4-F-Ph- ¹H NMR(500 MHz, CDCl₃) δ 8.10(d, 2H),7.26(d, 2H), 7.16(m, 2H) 6.80(s, 1H) 6.98(t, 2H), 6.80(s, 1H), 6.61(s,1H), 6.25(d, 1H), 5.55(d, 1H), 2.20(s, 3H), 1.65(m, 2H), 1.41(m, 2H),1.35(s, 3H), 0.91 (t, 3H, J = 7.30 Hz); m/z 474.52 2-30 4-O₂N-Ph CH₃—CH₂CH₃ 4-Cl-Ph- ¹H NMR(500 MHz, CDCl₃) δ 8.15(d, 2H, J = 9.05 Hz),7.28(d, 2H, J = 9.05 Hz), 7.10(d, 2H, J = 8.70 Hz), 6.80(s, 1H), 6.66(s,1H), 6.61 (d, 2H, J = 8.70 Hz), 6.27(d, 1H, J = 9.95 Hz), 5.57(d, 1H, J= 9.95 Hz), 2.20(s, 3H), 1.68(m, 2H), 1.60(m, 2H), 1.38(s, 3H), 0.92(t,3H, J = 7.30 Hz); m/z 490.99 2-31 4-O₂N-Ph CH₃ —CH₂CH₃ 4-NO₂-Ph- ¹HNMR(300 MHz, CDCl₃) δ 8.18(d, 4H, J = 9.2 Hz), 7.53(d, 2H, J = 9.2 Hz),7.40(br-s, 2H), 6.83(s, 1H), 6.67(s, 1H), 6.27(d, 1H, J = 9.9 Hz),5.59(d, 1H, J = 9.9 Hz), 2.22(s, 3H), 1.62-1.43(m, 4H), 1.38(s, 3H),0.93(m, 3H); m/z 501.55 2-32 4-O₂N-Ph CH₃ —CH₂CH₃ morpholine- ¹H NMR(300MHz, CDCl₃) δ 8.09(d, 2H, J = 8.2 Hz), 6.94(d, 2H, J = 8.2 Hz), 6.70(s,1H), 6.54(s, 1H), 6.25(d, 1H, J = 10.1 Hz), 5.55(d, 1H, J = 10.1 Hz),3.64(m, 4H), 3.32(m, 4H), 2.04(s, 3H), 1.68-1.57(m, 2H), 1.47-1.43(m,2H), 1.34(s, 3H), 0.91 (t, 3H, J = 7.2 Hz); m/z 450.55 2-33 4-O₂N-Ph CH₃—CH₂CH₃ (CH₂CH₃)₂— ¹H NMR(300 MHz, CDCl₃) δ 8.08(d, 2H, J = 9.0 Hz),7.05(d, 2H, J = 9.0 Hz), 6.68(s, 1H), 6.55(s, 1H), 6.23(d, 1H, J = 9.9Hz), 5.54(d, 1H, J = 9.9 Hz), 3.29(q, 4H, J = 7.2 Hz), 2.16(s, 3H),1.64-1.57(m, 4H), 1.33(s, 3H), 1.17(t, 3H, J = 7.2 Hz), 0.90(t, 6H, J =7.2 Hz); m/z 436.54 2-34 4-O₂N-Ph H —CH₂Ph Ph- m/z 504.59 2-35 4-O₂N-PhH —CH₂Ph 4-Me-Ph- m/z 518.63 2-36 4-O₂N-Ph H —CH₂Ph 4-MeO-Ph- m/z 534.622-37 4-O₂N-Ph H —CH₂Ph 4-F-Ph- m/z 522.59

TABLE 2e 2-38 4-O₂N-Ph H —CH₂Ph 4-Cl-Ph- m/z 539.02 2-39 4-O₂N-Ph H—CH₂Ph 4-NO₂-Ph- m/z 549.56 2-40 4-O₂N-Ph CH₃ —CH₂Ph Ph- m/z 518.63 2-414-O₂N-Ph CH₃ —CH₂Ph 4-Me-Ph- m/z 532.65 2-42 4-O₂N-Ph CH₃ —CH₂Ph4-MeO-Ph- m/z 548.65 2-43 4-O₂N-Ph CH₃ —CH₂Ph 4-F-Ph- m/z 536.64 2-444-O₂N-Ph CH₃ —CH₂Ph 4-Cl-Ph- m/z 553.07 2-45 4-O₂N-Ph CH₃ —CH₂Ph4-NO₂-Ph- m/z 563.63 2-46 4-O₂N-Ph CH₃ —CH₂Ph Ph-piperazine- m/z 587.742-47 4-O₂N-Ph CH₃ —CH₂Ph 2-MeO-Ph- m/z 617.76 piperazine- 2-48 4-O₂N-PhCH₃ —CH₂Ph 3-MeO-Ph- m/z 617.76 piperazine- 2-49 4-O₂N-Ph CH₃ —CH₂Ph4-F-Ph- m/z 605.72 piperazine- 2-50 4-O₂N-Ph CH₃ —CH₂Ph 4-Cl-Ph- m/z Z622.17 piperazine- 2-51 4-O₂N-Ph CH₃ —CH₂Ph 4-Me-Ph- m/z 601.76piperazine- 2-52 4-O₂N-Ph CH₃ Ph Ph- m/z 490.5

The following Examples are given for the purpose of illustration only ofseveral methods for preparing a formulation comprising the inventivecompound as an active ingredient, but they should be construed aslimiting the scope of the present invention.

Formulation 1: Tablet (Direct Pressurization)

After sieving 5.0 mg of the active ingredient, it was mixed with 14.1 mgof lactose, 0.8 mg of crospovidone USNF and 0.1 mg of magnesiumstearate, and the mixture was subjected to direct pressurization to beformulated into a tablet.

Formulation 2: Tablet (Wet Granulation)

After sieving 5.0 mg of the active ingredient, it was mixed with 16.0 mgof lactose and 4.0 mg of starch. After 0.3 mg of Polysorbate 80 wasdissolved in pure water, the proper amount of the resulting solution wasadded to the mixture and subjected to granulation. After drying, thegranules were sieved and mixed with 2.7 mg of colloidal silicon dioxideand 2.0 mg of magnesium stearate. The resulting granules were subjectedto pressurization to be formulated into a tablet.

Formulation 3: Powder and Capsule

After sieving 5.0 mg of the active ingredient, it was mixed with 14.8 mgof lactose, 10.0 mg of polyvinyl pyrrolidone and 0.2 mg of magnesiumstearate. The mixture was filled into a firm No. 5 gelatin capsule usinga suitable device.

Formulation 4: Injection

An injection was prepared by mixing 100 mg of the active ingredient, 180mg of mannitol, 26 mg of Na₂HPO₄.12H₂O and 2974 mg of distilled water.

Experimental Example Biological Assay Experimental Example 1 Experimentfor Antagonistic Activity on TGF-β Receptor

During the damage of hepatocytes which is a critical step for inducinghepatic fibrosis/liver cirrhosis, TGF-β cytokine produced and secretedby inflammatory cells and Kupffer cells induces the proliferation anddifferentiation of hepatic stellate cells, which results in theover-production and accumulation of an extracellular matrix such ascollagen. Therefore, it is possible to develop a compound capable ofinhibiting the proliferation and differentiation of hepatic stellatecells and suppressing a chemotactic mechanism of inflammatory cellsthrough the inhibition of TGF-β function as a therapeutic agent fortreating hepatic fibrosis/liver cirrhosis.

The present inventors have carried out an experiment for antagonisticactivity on TGF-β receptor to screen a compound capable of blocking aTGF-β-mediated intracellular signal transduction system by competitivelyinhibiting the binding between TGF-β receptor and its endogenous ligan,TGF-β. TGF-β dissolved in a sodium carbonate solution was added to eachwell of a well plate and the well plate was incubated at 4° C. overnightto attach TGF-β to the surface of the well. The purified biotin-TGF-βwas dissolved in a Tris-HCl buffer, added to the well together with acompound to be experimented, and then, the mixture was kept at roomtemperature for 1 hour to induce a binding reaction between TGF-β or andbiotin-TGF-β. After each well was washed with PBS-0.05% Tween 20solution (PBST buffer), HRP-conjugated streptavidin was added thereto,and the well plate was kept at room temperature for 1 hour. Each wellwas washed with the PBST buffer, a TMB solution as a HRP substrate wasadded thereto, and then, the well plate was kept at room temperature for20 min to develop a color. The reaction was stopped by adding an equalvolume of 1 M phosphoric acid solution. About 5 min after the reactionwas stopped, the absorbance of each well was measured at 450 nm of ameasurement wavelength and 540 nm of a correction wavelength. As aresult, it has been found that the benzopyran derivative in accordancewith the present invention shows antagonistic activity on TGF-βreceptor. The results of some compounds showing more than 50% of highreceptor antagonistic activity are illustrated in FIGS. 1 and 2,respectively. Since it has been hypothesized from the above results thatthe compounds showing antagonistic activity on TGF-β receptor are alsocapable of inhibiting the progression of fibrosis in hepatic stellatecells, the following experiments have been conducted.

Experimental Example 2 Experiment on Inhibitory Activity in CollagenSynthesis

Hepatic fibrosis is a procedure that collagen is accumulated byincreasing the synthesis of collagen and decreasing the degradationthereof due to the proliferation and activation of hepatic stellatecells. Accordingly, as hepatic fibrosis progresses, the activatedhepatic stellate cells stimulate the synthesis of collagen followed byincrease in the extracellular secretion of collagen thus synthesized,thereby confirming the inhibitory effect on hepatic fibrosis byobserving cytotoxicity and the inhibitory effect on collagen synthesisof hepatic stellate cells.

Human activated hepatic stellate cells, L190 cells were purchased fromJapan JCRB (Japanese Collection of Research Bioresources) cell linebank. L190 cell line was cultured at a 96-well plate using a Dulbecco'smodified Eagles medium (DMEM) supplemented with 10% fetal bovine serum(FBS) for 24 hrs. After replacing the medium with a fresh one withoutFBS, a test compound was treated thereto at various concentrations.Forty-eight hrs after the treatment of a test compound, MTS assay wasconducted using a CellTiter 96 non-radioactive cell proliferation assaykit (Promega) to measure cytotoxicity. Further, the amount of collagensynthesized within the culture solution was measured by a ELISA methodusing an anti-rabbit human collagen antibody (ABcam, England), and theresults are shown in FIGS. 3 and 4, respectively. Most of the benzopyranderivative compounds of the present invention showing antagonisticactivity on TGF-β receptor inhibited the proliferation of hepaticstellate cells, and also suppressed more than 50% of the synthesis anddegradation of collagen. Accordingly, it has been found that thesecompounds have the inhibitory effect on hepatic fibrosis by inhibitingthe proliferation of hepatic stellate cells that are abnormallyproliferated and activated followed by inducing the development ofhepatic fibrosis and suppressing the collagen synthesis and secretionfrom the cells.

Experimental Example 3 Inhibition of Collagen Gene Expression

Type 1 collagen as a major collagen involved in hepatic fibrosisconsists of α1 and α2 chains. Several transcription factors surroundinga collagen gene promoter take part in the expression of a collagenencoding gene, and generally, the amount of collagen expressed isdirectly proportional to the transcription efficiency of collagen.Therefore, it is capable of measuring a degree of the inhibitory effecton hepatic fibrosis by observing the decrease in the collagen promoteractivity. The inhibitory effect on the collagen promoter activity inthis Experimental Example was measured as follows.

pCOL1A2-Luc plasmid was prepared by inserting 3.3 kb of a promoter ofType 1 collagen α2 chain (COL1A2) into pGL3 vector comprising aluciferase gene as a receptor gene. In order to correct transfectionefficiency, Herpes simplex virus thymidine kinase (HSV-TK) vectorcomprising a Renilla luciferase gene was prepared. The same L190 cellline as used in Experimental Example 2 was co-transfected withpCOL1A2-Luc plasmid and HSV-TK vector by using a Lipofectamine plusreagent (Life Science, USA). Twenty-four hours after the transfection,the culture medium was replaced with a fresh DMEM without FBS. At thistime, a test compound was simultaneously added thereto, the cells werecultured for 24 hrs, and then, subjected to lysis. The luciferaseactivity was measured by using a dual-luciferase assay kit (Promega).The ratio between the firefly luciferase activity and the Renillaluciferase activity thus obtained was calculated and the degree ofactivity inhibition was determined from the ratio. The results are shownin FIGS. 5 and 6, respectively. Similar to the results of ExperimentalExample 2, it has been found that the benzopyran derivative of thepresent invention inhibits the expression of collagen by reducing morethan 60% of Col1A2 promoter activity and suppressing the transcriptionof a collagen encoding gene.

Experimental Example 4 Prophylactic and Therapeutic Effect on HepaticFibrosis/Liver Cirrhosis

When hepatocytes are damaged in the procedure of hepatic fibrosis,Kupffer cells engulf the damaged cells and secrete various cytokines.These cytokines induce the proliferation and activation of hepaticstellate cells (HSCs). The activated hepatic stellate cells synthesizecollagen and accumulate it at an extracellular matrix. Finally, hepaticfibrosis is developed by thus continuously accumulated collagen at theextracellular matrix. Carbon tetrachloride (CCl₄) induces liver damagedue to the cell membrane destruction and necrosis through a series ofactions mediated by an oxidation reaction of free radicals that aregenerated by the metabolism of cytochrome P450 present withinhepatocytes. Accordingly, it is capable of examining the inhibitoryeffect on hepatic fibrosis by analyzing the decrease in the accumulationof extracellular matrix due to the administration of carbontetrachloride.

Sprague-dawley male rats (SD, 5-week old) were adapted to experimentalenvironment for 1 week and the rats having about 225 g of an averagebody weight were used in the following experiment. All the rats in eachgroup (n=12) were abdominally administered with olive oil containing 10%CCl₄ at a concentration of 0.1 mL/kg at a day's interval (threetimes/week) for 4 weeks, to stably induce chronic hepatic fibrosis. Atthis time, the experimental animal's survival rate was maintained at100%. A test compound was dissolved in 0.5% carboxymethylcellulose (CMC)and orally administered to the animals in each experiment group at aconcentration of 10 mg/kg, 50 mg/kg, and 200 mg/kg, respectively. Theexperiment compound's efficacy was determined by histopathologicalobservation of the liver tissue after necropsying the rats in eachexperiment group at 2-week (n=6) and 4-week (n=6) and fixing the livertissue extracted therefrom. The histopathological observation wascarried out by observing the liver tissues obtained from the perishedrats in the experiment group and necropsied rats in the control groupwith a naked eye, fixing them with 10% neutral formalin, preparingtissue slices according to a conventional paraffin embedding procedure,carrying out a series of H&E (Hematoxylin & Eosin) staining, Azanstaining and Toluidine blue staining, and then, observing them with amicroscopy. Each of the liver tissues obtained from the negativecontrol, the positive control induced hepatic fibrosis/liver cirrhosisby oral administration of CCl₄, and the experiment group administeredwith the inventive compound for 2-4 weeks after the oral administrationof CCl₄ was observed with Azan staining to analyze their hepaticfibrosis/liver cirrhosis inhibitory and therapeutic effects onconnective tissues, and the representative animal experiment results areshown in Table 3 and FIG. 7, respectively.

TABLE 3 Compound No. Group Control CCl₄ 1-53 1-54 1-52 1-49 LesionNormal Fibrosis Fibrosis Fibrosis Mild Fibrosis (Grade 2) (Grade <1)(Grade <1) inflammation (Grade 1) (Grade 0)

As described above, the novel benxopyran derivatives of the presentinvention shows antagonistic activity on TGF-β receptor, inhibitoryeffect on the collagen synthesis and inhibitory effect on livercirrhosis, and accordingly, they can be effectively used for developinga prophylactic and therapeutic agent for treating liver cirrhosisdisease.

1. A benzopyran derivative of Formula (1) or a pharmaceuticallyacceptable salt thereof:

wherein Y is S or N—R⁴; R¹ and R⁴ are independently C₁-C₂₀ alkyl, amine,substituted or unsubstituted phenyl, substituted or unsubstitutedbenzyl, isovaline(methylester), naphthyl, or phenyl-X-(wherein, X iscarbonyl, or C₁-C₆ alkyl), or R¹ and R⁴ are fused together with thenitrogen atom to which they are attached to form a heterocycle having a5- to 7-member ring; R² is hydrogen, or C₁-C₅ alkyl; R³ is hydrogen,C₁-C₅ alkyl, substituted or unsubstituted phenyl, or substituted orunsubstituted benzyl; and wherein the substituted phenyl or thesubstituted benzyl is phenyl or benzyl substituted with 1 to 4substituents selected from the group consisting of halogen, nitro,benzyloxy, C₁-C₅ alkyl, C₁-C₅ alkoxy, C₁-C₅ haloalkyl, and C₁-C₅alkylsulfanyl.
 2. The benzopyran derivative or a pharmaceuticallyacceptable salt thereof, wherein R¹ and R⁴ are independently C₁-C₂₀straight, branched and cyclic alkyl; amine; phenyl; phenyl replaced with1 to 4 substituents selected from the group consisting of halogen,nitro, benzyloxy, C₁-C₅ alkyl, C₁-C₅ alkoxy, C₁-C₅ haloalkyl, and C₁-C₅alkylsulfanyl; benzyl; benzyl replaced with halogen;isovaline(methylester); morphorino; naphthyl; or R¹ and R⁴ are fusedtogether with the nitrogen atom to which they are attached to formpiperidine, piperidine replaced with C₁-C₅ alkoxycarbonyl, piperazine,or piperazine replaced with phenyl; R² is hydrogen, or C₁-C₅ alkyl; andR³ is hydrogen, C₁-C₅ alkyl, phenyl, or benzyl.
 3. A method forpreparing the benzopyran derivative of claim 1 which comprises:synthesizing a thiourea-based benzopyran derivative of Formula (1a) byreacting 6-amino-2,2′-disubstituted-2H-chromen of Formula (2) with anisothiocyanate derivative of Formula (3); and synthesizing aguanidine-based benzopyran derivative of Formula (1b) by reacting thethiourea-based benzopyran derivative of Formula (1a) with an aminederivative of Formula (5):

wherein R¹, R², R³, and R⁴ are the same as defined in claim
 1. 4. Themethod of claim 3, wherein the unreacted isothiocyanate derivative ofFormula (3) is removed by filtering with a scavenger resin containingthe amine group of Formula (4):

wherein {circle around (P)} is a solid support in the form of a polymerselected from the group consisting of polystyrene,polystyrene-divinylbenzene, polymethacrylic acid-dimethylacrylamide andpolyhydroxy methacrylic acid.
 5. The method of claim 3, wherein theunreacted amine derivative of Formula (5) is removed by filtering with ascavenger resin containing the isocyanate group of Formula (6):

wherein {circle around (P)} is a solid support in the form of a polymerselected from the group consisting of polystyrene,polystyrene-divinylbenzene, polymethacrylic acid-dimethylacrylamide andpolyhydroxy methacrylic acid.
 6. A formulation for treating liverdisease which comprises the benzopyran derivative of Formula (1) or apharmaceutically acceptable salt thereof:

wherein Y, R¹, R², and R³ are the same as defined in claim
 1. 7. Aformulation for treating fibroplasia disease through antagonisticactivity on TGF-β receptor which comprises the benzopyran derivative ofFormula (1) or a pharmaceutically acceptable salt thereof:

wherein Y, R¹, R², and R³ are the same as defined in claim
 1. 8. Theformulation of claim 7, wherein the fibroplasia disease includes hepaticfibrosis, liver cirrhosis, pulmonary fibrosis, dermatosclerosis andglomerular fibrosis.